Mytilus edulis is one of the most abundant and widely distributed benthic invertebrate species found in the North Atlantic. The aim of this study was to test the applicability of hemocytes from Mytilus edulis for in vitro toxicity testing, and quantify the cytotoxic response following exposure of the hemocytes to selected PAHs (naphthalene, phenanthrene, pyrene, benzo(a)pyrene, 2,6-dimethylnaphthalene, 1,5-/1,7-dimethylphenanthrene). Cell viability was assessed with three methods; metabolic activity (measured as alamar blue reduction capacity), membrane integrity (measured as 5-carboxyfluorescein diacetate, acetoxymethyl ester reduction capacity), and intracellular glutathione level (measured as monochlorobimane binding capacity). The use of multiple cytotoxicity endpoints has the advantage of giving possible insight into the mechanism of toxicity.
In order to identify an optimal medium to maintain the viability of hemocytes in vitro, four media were assessed (Leibovitz (L-15), L-15 supplemented with antibiotics, RPMI 1640 and phosphate-buffered saline (PBS)). It was decided to culture the cells in L-15 supplemented with antibiotics to ensure the aseptic conditions. The hemocytes cultured in the different media maintained high viability after 24 hr, 48 hr, and 96 hr, as opposed with those of a 192-hr incubation period.
In this study, DMSO was used as a vehicle for PAHs; therefore the cytotoxicity of three DMSO concentrations was examined (0.2%, 0.8%, and 3.2%), in order to avoid its cytotoxic effects. The data revealed cytotoxic effect in the 3.2% DMSO treatment, while no cytotoxic effect was observed after 0.2% and 0.8% DMSO treatment.A general tendency of increasing toxicity with the exposure period was observed by the EC50 values determined for the tested PAHs. Comparable EC50 values for each time point (24 hr and 48 hr) were observed from the fluorescent indicator dyes. However, in some cases variable EC50 values were determined.
The possible toxic mechanism discussed for each PAH included the following; 1) the general membrane disruption (cell membrane and mitochondrial membrane) and loss of organelle due to the lipophilic properties of the compounds as in case of naphthalene, phenanthrene, and pyrene, 2) the biotransformation role in toxicity and formation of toxic metabolites and the lipophilicity were potential mechanisms proposed for benzo(a)pyrene, 3) the structure-toxicity relationship was conducted to the subsided PAHs mechanism. By ranking the PAHs cyototoxicity it was found that pyrene and benzo(a)pyrene are the most toxic compounds. This study established responses in the cell viability of the blue mussel hemocytes exposed to different PAHs.